Although protein synthesis occurs intracellularly, some proteins function outside the cell. These extra-cellular proteins are referred to as secreted proteins. Many of the secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form. These pre-proteins contain an appended amino terminal extension called a signal or leader sequence (or signal peptide). The signal sequence plays an essential role in transporting the appended peptide into and/or through limiting cellular membranes.
Signal sequence encoding DNA can be included in recombinant expression vectors. The use of such vectors now makes it possible to transform compatible host organisms so they will produce heterologous gene products. The host organisms are often bacteria since bacteria can be grown with relative ease in chemically defined media. Growth of the organisms is rapid and high product yields are possible. When suitable host bacteria are transformed to produce desired gene products, such gene products are often easier to detect and purify if they are secreted into the periplasmic space or into the growth medium. Secretion of desired gene products into the medium avoids the necessity of breaking up the host organisms in order to recover the product. In addition, some heterologous gene products have a toxic effect on the host organisms. When such heterologous gene products are secreted rather than being allowed to accumulate within the host, they are less likely to interfere with normal cellular functions.
In some instances, the signal sequence is cleaved proteolytically during or after secretion to yield a mature protein product that is dissociated from the limiting cellular membranes through which it passed. In other instances, although the signal sequence is also cleaved, the mature protein remains bound to the cell membrane because of further modifications which lead to covalent attachments of lipid to the amino termini of the mature protein. Escherichia coli lipoproteins are an example of such membrane-bound proteins; E. coli betalactamase is an example of a protein that is dissociated from the membrane following cleavage of the signal sequence, and as a result the mature beta-lactamase is secreted into the E. coli periplasmic space. Such secreted proteins are called exoproteins. Other proteins, however, such as Bacillus licheniformis beta-lactamase, which herein is also referred to as penicillinase, can be processed into two different mature protein forms, one of which is a secreted exoprotein form, the other of which is a membrane-bound lipoprotein form. In the lipoprotein form of penicillinase, the first 26 of the 34 amino acids of the penicillinase signal sequence appear to function as the actual transport portion of the signal peptide. Some or all of the remaining 8 amino acids appear to be involved, along with some of the carboxy-terminal amino acids in the transport portion of the signal peptide, in the formation of the lipoprotein form.
Although not entirely understood, the mechanism by which mature protein (lipoprotein form) becomes bound to a cell membrane appears to involve the formation of a lipid bond at a site located near or within the signal peptide. Attachment of the lipid appears to anchor the protein into the cellular membrane. Specific amino acid sequences in the region of the junction of the signal sequences and the mature protein capable of forming membrane-bound forms are believed to function as recognition sites for the cellular agents involved in the chemical modifications leading to the formation of the membrane-bound lipoproteins. These specific chemical modifications are thought to include covalent bonding of fatty acids to the signal sequence moieties.
The usefulness of signal sequences capable of promoting lipoprotein formation would in some cases be improved if the recognition sequences could be modified in a way that would increase the amount of unbound exoprotein that is secreted into the periplasmic space or into the growth medium. Such a modified signal sequence could of course also lead to a decrease in the amount of the membranebound lipoprotein that is formed.